Differential Internalization of Somatostatin in COS-7 Cells Transfected with SST₁and SST₂Receptor Subtypes: A Confocal Microscopic Study Using Novel Fluorescent Somatostatin Derivatives¹

Abstract

A growing body of evidence suggests that neuropeptide binding to G protein-linked receptors may result in internalization of receptor-ligand complexes, followed by intracellular mobilization and degradation of the ligand into its target cells. Because of discrepant results in the literature concerning the occurrence of such a mechanism for the tetradecapeptide somatostatin (SRIF), we have reinvestigated this question by comparing the binding and internalization of iodinated and fluorescent derivatives of the metabolically stable analog of SRIF, [D-Trp8]SRIF, in COS-7 cells transfected with complementary DNA encoding the sst1 or sst2A receptor subtype. A series of fluoresceinyl and Bodipy fluorescent derivatives of [D-Trp8]SRIF-14 was purified by HPLC, analyzed for purity by mass spectrometry, and tested for biological activity in a membrane binding assay. Of the six compounds tested, fluoresceinyl and Bodipy derivatives labeled in position alpha (fluo-SRIF) retained high affinity for SRIF receptors. COS-7 cells transfected with complementary DNA encoding either sst1 or sst2A receptors both displayed specific, high affinity binding of iodinated and fluo-SRIF. At 4 C, the labeling was confined to the cell surface in both cell types, as indicated by the fact that it was entirely removable by a hypertonic acid wash and assumed a pericellular distribution in the confocal microscope. At 37 C, the fate of specifically bound ligand varied markedly according to the type of receptor transfected. In cells encoding the sst1 receptor, approximately 20% of specifically bound ligand was recovered in the acid-resistant (i.e. intracellular) fraction. This fraction remained clustered at the periphery of the cell, suggesting that it was being sequestered either within or immediately beneath the plasma membrane. By contrast, in cells transfected with sst2A receptors, up to 75% of specifically bound ligand was recovered inside the cells, where it clustered into small endosome-like particles. These particles increased in size and moved toward the nucleus with time, suggestive of receptor-ligand complexes proceeding down the endocytic pathway. These results demonstrate that neuropeptides may be processed differently depending on the subtype of receptor expressed in their target cells and suggest that these different processing patterns may reflect different modes of sensitization/desensitization and recycling of the receptors, and thereby of transmembrane signaling.

Somatostatin Receptor Desensitization in NG108-15 Cells

“…Both recombinant sst 2A receptors (48,62,72,73) and endogenous sst 2 -like receptors (39) are internalized in response to agonist activation, although apart from agonist binding, the trigger for endocytosis is still unknown. In the present study, we have obtained circumstantial evidence for internalization as the mechanism underlying endogenous sst 2B receptor desensitization.…”

“…Endocytosis of receptors can be measured by the uptake of bound radiolabeled or fluorescent agonists into the cell, and both of these procedures have been used to study sst receptor internalization (39,72). We did seek to obtain direct evidence for sst receptor internalization in NG108-15 cells using both radiolabeled and fluorescently tagged SRIF analogues.…”

Ligand binding to somatostatin receptors induces receptor-specific oligomer formation in live cells

“…Fluorescent SST ligands were prepared by N-terminal conjugation of SST-14 to FITC and Texas red (TR) followed by HPLC separation (17,18). Radioligand binding studies were carried out by reacting [ 125 I-LTT] SST-28 for 30 min at 37°C with cell membrane protein as reported (8,9).…”

Expression, pharmacology, and functional role of somatostatin receptor subtypes 1 and 2 in human macrophages

“…Of all SRIF receptor subtypes, sst 2 is the one that was found to be the most efficiently internalized by SRIF analogs [36,55,65,66], including L-779,976 [65,67]. In contrast, the internalization of sst 1 was suggested to be speciesspecific [68], although the cloned human sst 1 seems to internalize poorly [69,70]. Taken together, our results do not support the hypothesis that the lack of effects of SRIF-14 (and cortistatin-14) on macrophage viability can be ascribable to the more efficient receptor down-regulation by SRIF-14 as compared with its synthetic analogs.…”